Method for monitoring hygiene

ABSTRACT

A test method and test device for hygiene monitoring of a test sample from a biological material or a surface with a biological material to detect the presence of phosphate and glucose as a measure of hygiene by employing a swab-type, puncturable-membrane test apparatus or a lateral-flow or capillary-flow test apparatus.

REFERENCE TO PRIOR APPLICATIONS

The present application claims the benefit of International ApplicationPCT/US01/24054, filed Aug. 1, 2001, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/222,365, filed Aug. 1, 2000;and U.S. Provisional Patent Application Ser. No. 60/228,369, filed Aug.28, 2000; and U.S. Provisional Patent Application Ser. No. 60/267,173,filed Feb. 8, 2001; the contents of each of these provisionalapplications is hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Cleanliness in industrial and health care settings is critical. Thesurfaces of equipment used for food handling, storage, or processing aremajor sources of microbial contamination. Such contamination can lead todecreased shelf life of products and, if pathogens are present,transmission of disease. Microbial colonies develop rapidly. Continuousmonitoring of surfaces, e.g., hygiene monitoring, can help protectagainst the spread of disease.

Historically, microbial culturing was used to determine the presence ofmicroorganisms. However, culturing is time consuming and, therefore, thenecessary “real time” feedback to sanitation and food preparationpersonnel is not available. As a result, food exposed to surfaces whichwere later found to contain potentially harmful microorganisms couldenter the food supply.

Lateral-flow chromatographic test strips have been used for a variety ofdiagnostic purposes. The test described herein utilizes a lateral-flowtest strip to provide a means for rapid, sensitive, user-friendly,hygiene monitoring of surfaces. Material swabbed from a surface can bedetected by reactions involving the pathways described herein.

Recent attention has focused on the problems of biofilms. Biofilms arecreated when microorganisms land on a surface and attach to itsmicroscopic cracks and crevices. Almost immediately, the organism beginsto produce a polysaccharide-like material which in hours acts as a glueto stick bacteria and viruses to the surface. Biofilms are moreresistant to routine sanitizing techniques than are their free-livingcounterparts. It is, therefore, critical to generate rapid results,preferably within a few minutes.

Food residues on surfaces are nutrients for rapid growth ofmicroorganisms and the potentially resultant biofilm. Such food residuesare also a source of cross-contamination to other food products laterexposed to the same contact surface. Therefore, proper hygienemonitoring of a surface should include detection of a broad range ofcontaminants, including both biofilms and residual food.

During the 1990s various rapid and efficient test methods andapparatuses were developed for the detection of contamination onsurfaces. Such methods do not detect microbes directly but insteaddetect markers, such as ATP, which are indicative of either the presenceof microbes or the existence of residual food contamination of asurface.

One such apparatus is the POCKETSWAB® (POCKETSWAB® is a registeredtrademark of Charm Sciences, Inc. of Lawrence, Mass.), which rapidly andefficiently detects ATP on surfaces. The POCKETSWAB® apparatus detectsATP through the reaction of luciferin and luciferase, which, in thepresence of ATP, emits light. Light emission is measured using aluminometer. It is desired, and the primary object of this invention, toprovide a rapid, visual test for hygiene monitoring, and thereby avoidthe need for a luminometer or other reader.

There are various tests available in the field which provide a rapid andvisual result, thereby reflecting the degree of surface cleanliness.Such tests are of interest for use in, for example, restaurants andsupermarkets, where an instrument for reading results would not beacceptable, either because of the large volume needed or because theycould not be secured, or lack ease of use.

One such test is marketed by Celsis International, PLC of Cambridge,United Kingdom, under the trademark Spotcheckä. The SPOTCHECK™ employs acyclic “comproproportionation” reaction to detect ATP (see U.S. Pat. No.6,043,047, issued Mar. 28, 2000, and PCT International Publication No.WO 00/36139, published Jun. 22, 2000).

One example of a method for detection of inorganic phosphate isdescribed by N. Conrath et al, “A novel enzyme sensor for thedetermination of inorganic phosphate”, Analytica Chimica Acta 309 (195)47–52 (1995), which is incorporated herein by this reference. Thatmethod, however, requires skilled laboratory personnel, is timeconsuming and requires equipment.

It is an object of this invention is to provide a broad-spectrum test torapidly monitor the hygiene of a surface by detecting a variety oforganic and inorganic materials, food residues and microorganisms.

SUMMARY OF THE INVENTION

A new and improved user friendly, broad spectrum, test apparatus, systemand method adapted to provide a visual determination of surfacecontamination is the object of the current invention. The purpose of thetest is to rapidly—within one minute—detect the presence of certainbiological materials that are indicators of improper or inadequatesanitation and cleanliness. Test results are qualitative; a positiveresult indicates presence of residue and need to clean.

The invention comprises a method for the rapid, colorimetricdetermination of hygiene monitoring of surface contamination, whichmethod comprises adding to a sample containing phosphate a phosphorylaseenzyme and a carbohydrate substrate to provide a reaction product offree glucose and phosphate bound to a saccharide; and detecting saidfree glucose in a colorimetric reaction as a measure of surfacecontamination. Phosphorylases are enzymes that help catalyze cleavage ofa bond by orthophosphate. In the case of maltose, maltose phosphorylasecleaves the carbon-oxygen linkage between glucose molecules to produceglucose and glucose-1-phosphate. That is, a phosphorylase enzyme such asmaltose phosphorylase, and carbohydrate substrate, such as maltose,react with phosphate from the sample to produce α-D-glucose andβ-D-glucose-1-phosphate.

The invention also comprises a method for the rapid, colorimetricdetermination of hygiene monitoring comprising adding to a samplecontaining phosphate maltose and maltose phosphorylase and detecting theamount of glucose or phosphate in a colorimetric reaction as a measureof surface contamination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reaction scheme that is utilized for general hygienemonitoring;

FIG. 2 is a reaction scheme that may be utilized when sensitivity toanimal tissue is of particular importance;

FIG. 3A is a schematic view of the VERICLEEN™ swab removed from the testdevice;

FIG. 3B is a side perspective, see-through view of the VERICLEEN™ swab;

FIG. 4 is a side perspective view of the VERICLEEN™ test strip withinthe optional blister package with the package covering pulled partiallyback to expose the wetting solution and capillary-flow test strip;

FIG. 5 is a side perspective view of the VERICLEEN™ test strip includinga space or gap to prevent diffusion of color reagents;

FIG. 6 is a side perspective view of the wetting-agent dispenserattached to a test strip container; and

FIG. 7 is perspective view of the VERICLEEN™ test strip.

DESCRIPTION OF THE EMBODIMENTS

The purpose of the rapid, one-step, assay test for hygiene monitoring isto detect rapidly (within one minute, preferably within 2 minutes) thepresence of biological material as an indicator of sanitation andcleanliness. The two materials detected, within the same single servicetest, are glucose and phosphate. Glucose is detected directly. Phosphateis detected indirectly by the reaction of maltose phosphorylase withmaltose to produce glucose.

Enzymes are added to the assay system to increase the assay spectrum byconverting biological material to glucose or phosphate. Materials arestablilized, purified to remove phosphate, adenosine triphosphate (ATP),adenosine diphosphate (ADP), glucose, and glucosidase, and placed in aPOCKETSWAB® or lateral-flow test strip format, both of which are singleassay formats that permit an easy testing format and rapid analysis.

Inorganic phosphate is ubiquitous in nature. It is involved in energymetabolism and activation reactions in plants, animals, andmicroorganisms. Inorganic phosphate is an important component in manycompounds and, as a major biological mineral, it is present alone insignificant amounts. Phosphates are used extensively in the foodindustry, including also as additives in dairy products, cereals, meats,and soft drinks.

Similarly, carbohydrates, including sugars, are present in and onbiological materials in significant amounts. For example, glucose ispresent in blood, fruits, and honey. Acid phosphatase is endogenous tocertain animal tissue, and alkaline phosphatase is present in feces. Thedetection of phosphate, phosphatase, and glucose are markers ofcontamination. Other carbohydrates, including sugars, can be convertedto glucose for detection. For example, lactose, a milk sugar, can beconverted to glucose by β-galactosidase. Sucrose, which is used inprocessed foods and is found in cane sugar, fruits, and as invert sugarin processed foods, can be converted to glucose by sucrase or invertase.

The test utilizes several reaction pathways for the detection ofbiological material. The primary detection pathway uses maltose andmaltose phosphorylase for the detection of phosphate. Phosphate presentin the sample reacts with maltose and maltose phosphorylase to convertmaltose into glucose and glucose-1-phosphate. The glucose formed isdetected colorimetrically by methods well known in the art. The testalso uses additional pathways to increase sensitivity. These additionalpathways result in increased formation of either or both phosphate andglucose.

The test strip consists of nitrocellulose adhesively bound to apolystyrene backing, with one end having a sample pad absorbent area influid-flow contact with the nitrocellulose, and the other end havingoptionally attached to it, or incorporated into it, a container of asurface-wetting reagent. The container of surface-wetting agent can alsobe incorporated into the test strip packaging. The wetting reagentcontainer can be in the form of, for example, a burstable pouch orpeel-top container.

Within the test strip are reagents providing biochemical pathways forthe conversion of common surface biological material to glucose orphosphate. Also located within the test strip are reagents for thecalorimetric detection of glucose. All reagents are stabilized, purifiedto remove phosphate, ATP, ADP, glucose, and glucosidase, and eithersprayed onto the lateral-flow test strip, incorporated into theabsorbent pad, or mixed with wetting solution.

Also within the test strip, preferably at the end opposite thesample-absorbing end, there can be included a positive control area foruse in comparison of the color change and to confirm that adequatesample flow occurred.

Various embodiments comprise reagents for three (optionally, four orfive or more) separate pathways.

Pathway 1:

A. Using apyrase to convert ADP and ATP to phosphate; and

B. Using maltose phosphorylase to convert phosphate and maltose toglucose and glucose-1-phosphate.

Phosphate can exist in free form on a surface or as phosphate in ADP orATP. The phosphate component of ATP/ADP can be liberated by apyrase.Apyrase contains ADPase and ATPase activity and, therefore, converts ATPor ADP to AMP and phosphate. The liberated phosphate and the endogenousphosphate are detected by the reaction of maltose with phosphate andmaltose phosphorylase to produce glucose and glucose-1-phosphate. Theglucose formed from the reaction of maltose with phosphate in thepresence of maltose phosphorylase is detected as described in Pathway 3.

Pathway 2: Sugar Conversion to Glucose

Carbohydrates, including sugars, are present in and on biologicalmaterials in significant amounts. Many sugars can be converted toglucose for detection. For example, lactose, a milk sugar, can beconverted to glucose by β-galactosidase. Sucrose, which is used inprocessed foods and is found in cane sugar, fruits, and as invert sugarin processed foods, can be converted to glucose by sucrase or invertase.The glucose formed by these reactions is detected as described inPathway 3.

Pathway 3: Detection of Glucose

Reactions for the colorimetric detection of glucose are well known inthe art. For example, β-D-glucose+O₂+H₂O are converted by glucoseoxidase to D-glucono-δ-lactone+H₂O₂. The hydrogen peroxide formed fromthe above reaction, combined with a peroxidase enzyme, such ashorseradish peroxidase, converts a colorless substrate to a dye that isreadily visible. A mutarotase may be included to enhance sensitivity byconverting α-D-glucose associated with phosphorylation to β-D-glucose toreact with the glucose oxidase.

Other Pathways:

A: Glucose-phosphate, such as glucose-1-phosphate formed by the reactionof maltose with phosphate and maltose phosphorylase, Pathway 1 B, orpresent otherwise in the sample, can be cleaved by phosphatase, forexample, acid phosphatase, to produce both glucose and phosphate.Providing phosphatase as a test reagent, therefore, increases testsensitivity.

B: In certain environments, detection of phosphatase is important.Glucose-phosphate, for example, glucose-6-phosphate, can be provide as atest reagent for detection of phosphatase. If phosphatase is present inthe sample, glucose-6-phosphate will be cleaved to produce glucose andphosphate for detection, as previously described.

Sensitivity

The following test results compare Charm Sciences, Inc.'s POCKETSWAB®,read on both Charm Sciences, Inc.'s LUMINATOR® and Firefly instruments,with this new one-step assay test, referred to as the VERICLEEN™ test(VERICLEEN™ is a trademark of Charm Sciences, Inc. of Lawrence, Mass.).The VERICLEEN™ swabs, which do not require a reader, compares favorablyto the POCKETSWAB®, which requires a reader.

Test Ingredients

Test results described herein were generated using the following testingredients incorporated into a single-service device similar to thatdescribed in U.S. Pat. No. 5,827,675, issued Oct. 27, 1998; and U.S.Pat. No. 5,965,453, issued Oct. 12, 1999 (TEST APPARATUS, SYSTEM ANDMETHOD FOR THE DETECTION OF TEST SAMPLES). It should be noted that priorto use, all reagents must be verified as free from glucose and phosphatecontamination; otherwise, they must be purified using dialysis,de-salting, enzyme treatment, or other appropriate method.

EXAMPLE 1

Substrate composition:

-   -   Maltose (approximately 470 μg)    -   EHSPT (approximately 125 μg)    -   4-aminoantipyrine (approximately 62 μg)

All 3 reagents are first lyophilized separately in a stabilizing buffer,then optionally combined in a cellulose tablet formation.

EXAMPLE 2

Enzyme composition:

-   -   Maltose phosphorylase (approximately 1.5 units)    -   Horseradish peroxidase (approximately 5 units)    -   Glucose oxidase (approximately 12.5 units)    -   β-galactosidase (approximately 1 unit)    -   Apyrase (approximately 1.7 units)

All enzymes are lyophilized separately, then optionally combined in acellulose tablet formulation.

EXAMPLE 3

Liquid niblet:

-   -   Sterile deionized water with 1 mM CaCl₂ and preservative.

Comparison of Charm Sciences, Inc.'s VERICLEEN™ vs. POCKETSWAB®

Sample Preparation:

Solids: includes fruits, vegetables, ice cream, meats, bread, and flour

-   -   20% extract: 2 grams food +8 mL deionized water; pound with        tissue masher for 30 seconds; let solids settle    -   prepare all dilutions from the 20% extract in deionized water

Liquids: includes orange juice, milk, eggs, and soda

-   -   product “as is” is 100% extract    -   prepare all dilutions from the 100% extract in deionized water

Assay:

-   -   1. Inject 50 μL extract directly into swab;    -   2. Activate swab device by twisting;    -   3. For PKS assay, count immediately on the Firefly analyzer; and    -   4. For VERICLEEN™, set time counting up; note time when purple        color appears.

Blank testing:

-   -   run swab devices without removing swab

POCKETSWAB ® Blanks VERICLEEN ™ Blanks Firefly RLU Reading 0 colordevelopment after 6 minutes 0 color development after 5.5 minutes 0color development after 6 minutes

TABLE 1 POCKETSWAB ® VERICLEEN ™ Fruits and Extract Firefly RLU Result(DNR = Vegtables Concentration Reading did not run) Celery   20%  265187DNR Celery   5%  74256 DNR Celery   1%  19918 DNR Celery  0.1%   2943purple in 30 seconds Cucumber   20%  270914 DNR Cucumber   5%  57434 DNRCucumber   1%  41790 purple in 20 seconds Cucumber  0.1%   2965 purplein 2.5 seconds  Carrot   20%  311245 purple in 15 seconds Carrot   5% 67449 purple in 30 seconds Carrot   1%  10318 DNR Carrot  0.1%   205purple in 60 seconds Lettuce   20%  130230 DNR Lettuce   5%  30816 DNRLettuce   1%   4689 purple in 30 seconds Lettuce  0.1%    0 purple in  2minutes Apple   20%  735903 DNR Apple   5%  162292 DNR Apple   1%  60205DNR Apple  0.1%   2747 purple in 15 seconds Orange   5% 3108565 DNR(fresh   1%  872245 DNR squeezed  0.1%  120521 DNR juice) 0.01%  23387purple in 40 seconds Orange   5% 3264303 DNR Juice   1%  819838 DNR(pasteurized)  0.1%  145470 DNR Orange Juice 0.01%   4961 purple in(pasteurized) 60 seconds

TABLE 2 POCKETSWAB ® VERICLEEN ™ Dairy Extract Firefly RLU Result (DNR =Products Concentration Reading did not run) Raw Milk   5% 50081 DNR RawMilk   1% 29572 DNR Raw Milk  0.1% 13732 purple in 45 seconds Raw Milk0.01%  1547 purple in  4 minutes Pasteurized   50% 213870  purple inMilk 45 seconds Pasteurized   5% 35594 pale purple in Milk  4 minutesPasteurized   1%  6074 DNR Milk Pasteurized  0.1%   0 DNR Milk Whole egg  5%  2714 DNR beaten Whole egg   1%   0 DNR beaten Whole egg  0.1%   0purple in beaten 30 seconds Whole egg 0.01%   0 purple in beaten 45seconds Soft serve   20% 47594 purple in ice cream 30 seconds Soft serve  5% 10241 purple in ice cream 45 seconds Soft serve   1%   0 purple inice cream  2 minutes Soft serve  0.1%   0 purple in ice cream 3.5minutes 

TABLE 3 POCKETSWAB ® VERICLEEN ™ Extract Firefly RLU Result (DNR = MeatsConcentration Reading did not run) Raw Ground  20%  279489 DNR Beef RawGround   5%  31154 DNR Beef Raw Ground   1%   3543 DNR Beef Raw Ground0.1%    0 purple in Beef 20 seconds Cooked  20%  831281 DNR Ground BeefCooked   5% 3629780 DNR Ground Beef Cooked   1% 4773347 purple in GroundBeef 30 seconds Cooked 0.1% 1305347 purple in Ground Beef  4 minutes Rawchicken  20%  457994 DNR breast Raw chicken   5%  43972 DNR breast Rawchicken   1%    0 purple in breast 60 seconds Raw chicken 0.1%    0purple in breast 1.5 seconds  Cooked  20%  694045 DNR chicken breastCooked   5% 3825860 purple in chicken breast 45 seconds Cooked   1%4457393 Negative at chicken breast  4 minutes Cooked 0.1% 1730583 DNRchicken breast

TABLE 4 POCKETSWAB ® VERICLEEN ™ Extract Firefly RLU Result (DNR =Grains Concentration Reading did not run) White Flour  20% DNR Whiteflour   5% purple in 20 seconds White flour   1% purple in 30 secondsWhite flour 0.1% purple in  2 minutes White bread  20% DNR White bread  5% DNR White bread   1% DNR White bread 0.1% purple in 50 seconds

TABLE 5 POCKETSWAB ® VERICLEEN ™ Extract Firefly RLU Result (DNR = SoftDrinks Concentration Reading did not run) Coke   5% 0 DNR Coke   1%purple in 30 seconds Coke  0.1% purple in 60 seconds Coke 0.01% palepurple at  4 minutes Sprite   5% 0 purple in 20 seconds Sprite   1% DNRSprite  0.1% purple in 60 seconds Sprite 0.01% DNR

The invention also comprises a method for increasing the above-describedhygiene monitoring test sensitivity to phosphate, through a cyclingreaction. Phosphatase, for example acid phosphatase, which is includedas a test reagent and may be present in the sample, cleaves boundphosphate, such as glucose-1-phosphate and glucose-6-phosphate into itscomponent, glucose and phosphate which both will be detected within theassay system. Glucose-1-phosphate and glucose-6-phosphate may be fromthe sample and also, in the case of glucose-1-phosphate, generated as areaction product of the carbohydrate substrate with phosphate in thepresence of an appropriate enzyme as previously described. The reactionproduct glucose-1-phosphate is broken down by phosphatase to producemore glucose for the color change reaction and more phosphate to combinewith the carbohydrate substrate as previously described.

The invention also comprises a way to release phosphate and glucose fromvarious commonly found surface contaminants such as adenosinetriphosphate (ATP), adenosine diphosphate (ADP), biofilms,carbohydrates, microbes, and phosphatase. A method for releasingphosphate and glucose from such contaminants includes using enzymes suchas phosphatase, for example apyrase and glucose phosphatase, andenzymes, such as hydrolytic enzymes, to breakdown carbohydratescomprised of more than one saccharide (complex carbohydrates) to releasethe glucose component, for example the breakdown of lactose byβ-galactosidase, the breakdown of sucrose by invertase and the breakdownof starch by amylase. A method for releasing various molecules such asATP, ADP and glucose from microbes includes the use of lysing reagents.A mutarotase may be included to enhance sensitivity by convertingα-D-glucose associated with phosphorylation to β-D-glucose to react withthe glucose oxidase.

The presence of acid phosphatase on a surface is indicative ofcontamination with certain animal tissue. A separate embodiment of theinvention includes additional way to detect phosphatase. Such methodsmay be included within the assay system in circumstances where detectionof phosphatase is relatively more important than increasing testsensitivity to phosphate, for example when detection of animal tissue,such as muscle or blood, is relatively more important compared to thegeneral increase sensitivity to phosphate. However, the above-describedmethods for increasing test sensitivity to phosphate, by providingphosphatase as a reagent, cannot practically be combined with themethods for detecting phosphatase in a sample. Methods for detectingphosphatase include providing glucose-6-phosphate or glucose-1-phosphateas a test reagent. Phosphatase present in the sample will cleave, forexample glucose-6-phosphate, into is components glucose and phosphate.

The invention involves a method for the rapid, colorimetricdetermination of hygiene monitoring of surface contamination, whichmethod comprises: adding a phosphorylase enzyme and carbohydratesubstrate to a sample containing phosphate to create a reaction productof free glucose and phosphate bound to a saccharide; cleaving freephosphate from said bound phosphate; cleaving free glucose from complexcarbohydrates; releasing or extracting intracellular phosphate; andchanging the interconversion of α-D-glucose to β-D-glucose, wherein freeglucose is detected in a colorimetric reaction as a measure of surfacecontamination.

The invention also involves a method for the rapid, colorimetricdetermination of hygiene monitoring of surface contamination includingcontamination with phosphate and phosphatase, which method comprises:adding a phosphorylase enzyme and a carbohydrate substrate to a samplecontaining phosphate to create a reaction product of free glucose andphosphate bound to a saccharide; cleaving free phosphate from boundphosphate; cleaving free glucose from complex carbohydrates; releasingintracellular phosphate; and catalyzing the interconversion ofα-D-glucose to β-D-glucose, wherein free glucose is detected in acolorimetric reaction as a measure of surface contamination.

The invention also comprises a device for rapid, colorimetric hygienemonitoring, which test device comprises: a capillary-flow test striphaving two ends, the test strip includes at a first end, a liquid sampleabsorbing material; reagents for conversion of biological material toglucose, such as by providing multiple biochemical pathways; andreagents for the colorimetric detection of glucose, wherein a liquidsample from a material is absorbed onto the first end and flows bycapillary action to contact reagents providing biochemical pathways forconversion of biological material to glucose and colorimetric detectionof glucose.

The invention also comprises a device for rapid, colorimetric hygienemonitoring having one or more reaction zones, included on or in acapillary membrane, comprising reagents for conversion of biologicalmaterials to glucose and phosphate and reagents which, in the presenceof phosphate and an appropriate enzyme, convert certain biologicalmaterials to glucose; and reagents for the colorimetric detection ofglucose, wherein a liquid sample from a material is absorbed onto thefirst end and flows by capillary action through the test stripcontaining reagents, which produces a visually detectable readout in thepresence of glucose.

The invention also comprises a device for hygiene monitoring, bydetecting biological material in a test sample from or on a material,which test device comprises: a swab to collect the test sample and aniblet comprising separate, swab-puncturable, membrane compartments forone or more reagents and a solution; an enzyme reagent composition,preferably in tablet form, comprising glucose oxidase, horseradishperoxidase, maltose phosphorylase, apyrase, β-galactosidase andphosphatase; a substrate reagent composition, preferably in tablet form,comprising 4-aminoantipyrene, mutarotase, EHSPT (the Trinder's reagentTOOS), and maltose; and a liquid reagent comprising a buffering solutionand lysing solution, wherein the sample is contacted sequentially withthe liquid reagent, enzyme reagent, and substrate reagent and a colorchange or lack thereof is observed as a measure of hygiene.

The test apparatus of certain embodiments of the invention is in theformat of the POCKETSWAB®, which is described in U.S. Pat. No.5,965,453, issued Oct. 12, 1999 (TEST APPARATUS, SYSTEM AND METHOD FORTHE DETECTION OF TEST SAMPLES); U.S. Pat. No. 5,985,675, issued Nov. 16,1999 (TEST DEVICE FOR DETECTION OF AN ANALYTE); and U.S. Pat. No.6,180,395, issued Jan. 30, 2001 (REAGENT CHAMBER FOR TEST APPARATUS ANDTEST APPARATUS), which are incorporated herein in their entirety. Inthese embodiments, the apparatus incorporates a foam-tipped, or otherabsorbent-type swab or wand for sample uptake from the surface to bemonitored. The swab may be premoistened with a wetting solution.

After sample uptake onto the swab, the swab is used to puncture a seriesof “niblets” releasing and activating the necessary reagents. The term“niblet” refers to a reagent chamber in the form of a cylindercontaining reagents and sealed on both ends with a probe puncturablemembrane.

In certain embodiments, the first niblet to contact the swab includescofactors and buffering compounds to optimize subsequent reactions andantimicrobial or antifungal substance to prevent contamination. In otherembodiments, the first niblet may additionally contain bacterial lysingreagents.

After the swab is contacted with the first reagent niblet, the swab iscontacted with the material in the second niblet by, preferably,puncturing the membranes separating the first and second niblet, therebycausing reagents from the first niblet to flow into and mix withreagents in the second niblet. The second niblet contains one or morereagents, preferably tablets, containing enzymes, substrates, andbiochemicals. Certain embodiments include two tablets in the secondniblet, generally referred to as the “substrate tablet” and the “enzymetablet.”

In some embodiments, the substrate reagent composition includes maltose,glucose-phosphates, such as glucose-1-phosphate or glucose-6-phosphate,TOOS and 4-aminoantipyrine, and the enzyme reagent includes maltosephosphorylase, horseradish peroxidase, glucose oxidase, mutarotase,β-galactosidase and apyrase. In other embodiments, enzymes forconverting other carbohydrates to glucose, for example, amylase, sucrase(invertase) may be included.

In another embodiment, glucose-6-phosphate is removed from the substratereagent. Such an embodiment will be less sensitive to samplephosphatase. Instead, phosphatase is added to the enzyme reagent orsubstrate reagent. The added phosphatase will cleave theglucose1-phosphate reaction product of maltose and phosphate to produceadditional phosphate and glucose, thereby enhancing test sensitivity tophosphate through the additional cycling of the phosphate group, fromthe glucose-1-phosphate, to be combined with maltose and maltosephosphorylase. It will be appreciated that although the reagents ofparticular embodiments are described as “tablets” or “liquid,” thereagents can be used in, and applied to, the test device in a variety offorms, which forms are prepared separately or in combination, includingsolid, liquid, powder, freeze-dried, emulsion, suspension, tablet, orany other form known to those skilled in the art.

The test apparatus of certain other embodiments of the invention is inthe format of a capillary-flow test strip. The strip consists of acapillary-flow capillary membrane, for example, nitrocellulose membrane,adhesively bound to a backing, for example a polystyrene backing, withone end having a sample pad absorbent area in fluid flow contact withthe membrane. Optionally, at the sample pad end, or the other end, thestrip could have attached, or incorporated into the packaging, acontainer of a surface wetting reagent. The container of surface-wettingagent may include a wetting solution. The wetting solution container canbe in the form of, for example, a burstable pouch or peel-top containerwithin the test strip packaging. Alternatively, the surface-wettingsolution can be included in a bulk container, for example, a squeezable,plastic, dispenser bottle or spray bottle, which is attached at the endopposite the dispensing end, to a container within which capillary-flowstrips are placed. The wetting solution should, optimally, be pHneutral, food compatible and non-interfering. The capillary-flow stripcontainer is formed of a light-blocking material to protect the teststrips. Applied to the test strip capillary membrane are reagents forthe production of glucose and phosphate from common surface biologicalmaterial and reagents for the colorimetric detection of glucose.

In one example of the capillary-flow strip, reagents are provided in twozones. The two reagent zones are applied to the membrane portion of thetest strip in fluid flow contact with the absorbent pad. Optionally, thereagents can be applied by spraying onto the strip. In one embodiment,the first reagent zone contains a carbohydrate substrate, for example,maltose and glucose-6-phosphate. In an alternative embodiment, the firstreagent zone contains the carbohydrate substrate and a phosphatase, forexample, acid phosphatase. The second reagent zone contains reagents forthe colorimetric detection of glucose, for example, TOOS, glucoseoxidase, mutarotase, horseradish peroxidase and 4-aminoantipyrene. Alsoincluded within the second reagent zone are enzymes, such as maltosephosphorylase and apyrase and one or more hydrolytic enzymes, such asβ-galactosidase.

The reagents in this device are able to flow in the membrane reactingwith the sample and each other. Sufficient capillary-flow space isincluded beyond the second reagent zone for color reagents to changecolor in the presence of a target material and provide an easily read,visible result. The space beyond the second reagent zone is sufficientlysmall to create a defined area for color change to occur. Color changebegins at the end of the capillary membrane and spreads back. Providingspace between the second reagent zone and the end of the capillarymembrane permits a wider, more visible line to develop. At the end ofthe capillary membrane is an air gap to prevent reagent flow beyond theend of said membrane, a wetting solution for solubilizing surface foodresidue and penetrating biofilm.

All reagents are stabilized, purified to remove phosphates, glucose andmaltose degrading enzymes, for example, glucosidase, and either sprayedonto the capillary-flow membrane or incorporated into the absorbent pad.

The test apparatus system and method will be described for the purposesof illustration only in connection with a series of illustrative testapparatus and test method employing various test apparatus. However, itis recognized that those persons skilled in the art may make variousmodifications, changes, additions, and improvements to the testapparatus, system and methods without departing from the spirit andscope of the invention.

EXAMPLE 4

The lateral or capillary-flow strip consists of a nitrocellulosemembrane onto which two regions, zones, or lines of reagents have beenapplied using appropriate manufacturing equipment, such as that made byBioDot, Inc. Optionally, the membrane is overlapped with an absorbentcellulose paper pad. One region consists of all enzymes needed for thecolor reaction, the enzymes for release of phosphate and glucose, plusthe two substrates necessary for color development. Prior to use, allenzymes must be purified to remove any sugar or phosphate contamination,for example, by desalting. The second line contains a preparation ofglucose-free maltose and purified acid phosphatase. The nitrocelluloseis mounted onto a polystyrene backing material and, optionally, packagedinto a plastic, blister-type device. If packaged into a blister-typedevice the device, optionally, contains a bubble to which surfacewetting agent is added; the bubble may be sealed with peelable foil.

The reagents applied to the capillary membrane are the same as thoseused as reagents or tablets for the swab-type assay. However, the amountof each component required per test is significantly lower for allreagents. The approximate amount of each reagent and its correspondingamount used in the swab test are listed in Table 6, in an embodimentincluding glucose-6-phosphate and Table 7 in an embodiment includingacid phosphatase.

Results generated with the embodiment described in Table 6 are listed inAppendix 1.

TABLE 6 Amount per Test in Amount per Test Reagent Capillary-FlowMembrane in Swab Test TOOS 930 ng 125 mg 4-aminoantipyrine 460 ng 63 mgMaltose 2 mg 470 mg Maltose phosphorylase 0.06 units 1.5 units Glucoseoxidase 0.5 units 12.5 units Horseradish peroxidase 0.2 units 5 unitsβ-galactosidase 0.04 units 0.8 units Glucose-6-phosphate 10 mg 100 mgApyrase 0.2 units 1.5 unitsAppendix 1

Test Data

I. Assay in Which Solution of Food Product is Allowed to Air Dry ontoCountertop then Rehydrated with Wetting Solution and Wiped with TestStrip

Time to Color Food Concentration Change (s) Beef 2% 16, 15 Chicken 5%10, 7  Fish 5% 20, 23 Ketchup 1% 3, 2 Milk 10%  35, 37 Orange juice 2%4, 3 Egg 2% 4, 5II. Assay in Which Wet Solution of Food Product is Wiped from PlasticSurface with Test Strip

Time to Color Food Concentration Change (s) Beef 2%  8, 10 Chicken 5% 8,7 Fish 5% 9, 8 Ketchup 1% 2, 2 Milk 10%  30, 32 Orange juice 2% 2, 3 Egg2% 5, 5

TABLE 7 Amount per Test in Amount per Test Component Capillary-FlowMembrane in Swab Test Glucose oxidase 0.7 units 12.5 units Horseradishperoxidase 0.3 units 5 units β-galactosidase 0.06 units 1 units Maltosephosphorylase 0.09 units 3 units Apyrase 0.3 units 5 units Acidphosphatase 0.01 units 0.1 units Maltose 4 mg 1400 mg 4-aminoantipyrene700 ng 63 mg N-ethyl-N-(2-hydroxy- 1400 ng 125 mg 3-sulfopropyl)-m-toluidine

Materials are stabilized, purified to remove phosphate, ATP, ADP,glucose and glucosidase, and placed in a POCKETSWAB® or capillary-flowmembrane format, both of which are single assay formats that permit aneasy testing and rapid analysis.

FIG. 1 illustrates the general use reaction scheme of the invention,which is particularly useful when testing for a wide range ofcontaminants. Maltose reacts with phosphate from the sample in thepresence of maltose phosphorylase to form α-D-glucose andβ-D-glucose-1-phosphate. β-D-glucose forms color when combined withcertain well-known reagents. α-D-glucose naturally converts toβ-D-glucose. The rate of natural conversion can be enhanced bymutarotase. The β-D-glucose-1-phosphate in the presence of acidphosphatase (as a test reagent) is broken down into phosphate andβ-D-glucose (Phosphatase from the sample will also break downβ-D-glucose-1-phosphate.). The glucose enters into the color reactionamplifying the color from the first glucose, and the phosphate reactswith more maltose (a test reagent) to generate more glucose and,ultimately, more phosphate and more color until the maltose reagent isdepleted, or the reaction is otherwise inhibited. The above-describedcycling of phosphate produces a sensitive test for phosphate in thesample. The reaction scheme also includes examples of conversion ofcommon biological material to glucose and phosphate. Lysing reagentsrelease ATP and ADP from microbes and apyrase cleaves the phosphate.Carbohydrates are hydrolized to release glucose for detection.

FIG. 2 illustrates the reaction scheme particularly useful for detectingcontamination from raw or partially-cooked meat products.Glucose-6-phosphate (or alternatively glucose-1-phosphate) is added as areagent to include an additional source of phosphate and glucose whenacid phosphatase is present in the sample. In the embodiment representedby said reaction scheme, acid phosphatase is not a reagent and is,instead, supplied from the sample. If acid phosphatase is not present,the glucose-6-phosphate does not enter the reaction, and only phosphatefrom the sample generates glucose. Color is generated by the glucose.

FIGS. 3A & B illustrate the invention in the format of the swab-typedevice. In use of the swab-type device of the invention, the swab 1 isremoved from the body 3, by gripping the swab handle 2, and a 4″×4″surface, for example, a food contact surface, is swabbed using thepre-moistened swab 1. The swab 1 is then reinserted into the body 3 andscrewed longitudinally through the covering 9 of the microtube test unit4 and through the covering 10 of the liquid reagent niblet 5 and intothe tablet niblet 6 containing an enzyme tablet 7 and a substrate tablet8. The liquid released to the bottom of the microtube test unit 4 turnspurple within 60 seconds if the surface is “dirty,” for example, it hasfood residue contamination.

FIG. 4 illustrates the invention in the capillary-flow strip-type devicepackaged within a blister package. In use of the capillary-flow striptest-type device of the invention, the blister package cover 12 ispeeled back, and the surface-wetting solution is released from thecontainer 11 onto the surface to be monitored. The user continues topeel back the covering 12 to expose the test strip 13.

FIG. 5 is a side perspective view of the VERICLEEN™ test strip. Whilehandling area 14 the absorbent pad 15 is used to swab the surface to bemonitored. The sample is absorbed onto sample pad 15 and flows bycapillary action through the first reagent zone 19, to the secondreagent zone 20 (Reagent zones 19 and 20 are depicted forillustration.). In the preferred embodiment, reagent zones 19 and 20 areslightly visible or invisible. Sample flow stops at the end of thecapillary membrane 18. Positive results are reflected in color changezone 17. Color change begins to form at the end of the capillarymembrane 18 and spreads back. A gap 21 is included sufficiently wide toprevent unwanted diffusion of color into the cover 22 of the backing 23,which cover 22 and backing 23 combined make up the handling area 14.Line development at the top of the capillary membrane, withinapproximately one minute, indicates presence of residue and need toclean. All valid tests will change color after approximately fiveminutes. Reagent zones 19 and 20 contain reagents depicted in thereaction scheme of either FIG. 1 or FIG. 2, depending on therequirements of the user.

FIG. 6 is a side perspective view of the wetting-agent dispenser 24attached to a light-blocking test strip container 25.

FIG. 7 illustrates an embodiment of the present invention containing astrip of nitrocellulose adhesively bound to a polystyrene support.Various reagents are sprayed and dried onto the nitrocellulose in aseries of discreet zones. FIG. 7 contains seven such spaced-apart zonesas follows:

Zone 1, 48, is comprised of apyrase and β-galactosidase. Apyrase cleavesany ATP/ADP liberating phosphate that will flow in the solution alongthe test strip to Zone 2. β-galactosidase converts lactose to glucose.Zone 2, 46, comprises maltose for reaction with phosphate, in thepresence of the maltose phosphorylase to produce glucose. Zone 3, 44, iscomprised of maltose phosphorylase. Zone 4, 42, is comprised of glucoseoxidase for oxidation of glucose in a reaction that yields peroxide.Zone 5, 40, is comprised of EHSPT[N-ethyl-N-(2-hydroxy-3-sulfo-propyl)-n-toluidine], commonly referred toas TOOS; Zone 6, 30, is comprised of 4-aminoantipyrine; and Zone 7, 36,is comprised of horseradish peroxidase. Zones 5, 6, and 7, 40, 30, and36, respectively comprise reagents which, in the presence of peroxide,produce a color precipitate which can be observed visually.

It is also possible to combine the seven zones described above intofewer zones, for example, three zones, containing multiple reagents perzone.

In operation, the wetting reagent is released onto the surface to betested or absorbed onto the absorbent pad 15. Holding the strip at thefinger-hold area 32, the sample pad 15 is used to swab the surface to betested. Wetting the surface will suspend in the liquid some of thematerial to be detected. Material to be detected will be absorbed by thestrip upon swabbing the wet surface. The liquid absorbed onto the stripwill then flow laterally on the strip. Upon contact with afluid-analyte, the reactants in the zones are resolubilized and reactwith the analyte(s) in the sample as described above. Color formation inthe horseradish peroxidase 36 reaction zone indicates a positive sample,while no color indicates a clean sample.

1. A method for rapidly monitoring the hygiene of an inanimate surface desired to be free of the presence of biological contamination, which method comprises the steps of: a) obtaining a sample from said surface; b) adding to said sample one or more enzymes, said one or more enzymes convert a carbohydrate that contains glucose to glucose; and c) detecting the presence or absence of glucose that has been enzymatically derived in step b from said sample, wherein the presence of said derived glucose from said sample is an indicator of said contamination on said surface and wherein less than 10 minutes elapses between step b and step c.
 2. The method of claim 1, wherein said carbohydrate comprises one or more sugars.
 3. The method of claim 1, wherein said carbohydrate comprises one or more complex carbohydrates.
 4. The method of claim 1, wherein said carbohydrate comprises lactose.
 5. The method of claim 1, wherein said enzyme comprises a hydrolytic enzyme.
 6. The method of claim 5, wherein said enzyme comprises invertase.
 7. The method of claim 5, wherein said enzyme comprises amylase.
 8. The method of claim 5, wherein said enzyme comprises cellulase.
 9. The method of claim 5, wherein said enzyme comprises β-galactosidase.
 10. The method of claim 5, wherein said enzyme comprises sucrase.
 11. The method of claim 1, wherein said detecting comprises adding to said sample a reagent for the colorimetric detection of said glucose.
 12. The method of claim 11, wherein said reagent for the colorimetric detection of glucose comprises glucose oxidase.
 13. The method of claim 11, wherein said reagent for the colorimetric detection of glucose comprises peroxidase.
 14. The method of claim 1 comprising catalyzing the interconversion of α-D-glucose to β-D-glucose.
 15. The method of claim 1, comprising using a swab to obtain the sample from said surface.
 16. The method of claim 1, further comprising introducing the sample to the enzyme by applying the sample to a capillary-flow membrane, said membrane including the reagent.
 17. The method of claim 1, further comprising adding to said sample a phosphorylase enzyme and a carbohydrate substrate to produce, in the presence of a phosphate in said sample, a reaction product of glucose and a phosphate-bound saccharide reaction product.
 18. The method of claim 17, wherein the phosphorylase enzyme comprises maltose phosphorylase, and the carbohydrate substrate comprises maltose.
 19. The method of claim 17, further comprising cleaving inorganic phosphate from an organic molecule containing phosphate.
 20. The method of claim 17, wherein said cleaving comprises adding a phosphatase enzyme.
 21. The method of claim 20, wherein the phosphate-bound saccharide reaction product is glucose-1-phosphate, and wherein the phosphatase enzyme cleaves phosphate from said glucose-1-phosphate.
 22. The method of claim 20, wherein the phosphatase enzyme comprises apyrase.
 23. The method of claim 20, wherein the phosphatase enzyme comprises acid phosphatase.
 24. The method of claim 20, wherein the phosphatase enzyme comprises glucose phosphatase.
 25. The method of claim 20, wherein said phosphatase enzyme comprises ATP hydrolase.
 26. The method of claim 20, wherein the phosphatase enzyme comprises alkaline phosphatase.
 27. The method of claim 1, further comprising releasing intracellular phosphate from a biological cell in said sample.
 28. The method of claim 27, wherein said intracellular phosphate is released by a lysing reagent.
 29. The method of claim 1, further comprising adding glucose-phosphate to a sample suspected of containing phosphatase, to produce a reaction product of glucose and phosphate.
 30. The method of claim 29, wherein the glucose-phosphate is glucose-6-phosphate.
 31. The method of claim 1, which method comprises introducing the sample to the reagents through one or more membrane-puncturable niblets.
 32. The method of claim 1, which method comprises introducing the sample onto a capillary-flow membrane containing reagents for production of glucose and components for colorimetric detection of glucose.
 33. The method of claim 32, wherein the reagents are not immobilized on the membrane and which method further comprises preventing flow of reagents after test completion so as to provide a defined area for detecting a color change reaction.
 34. The method of claim 33, wherein a wetting agent is included in a sealed area within packaging of a test strip, and said wetting agent is applied to a surface sample prior to introducing said surface sample onto the test strip.
 35. The method of claim 1, wherein said sample is a liquid obtained from a surface.
 36. The method of claim 1, wherein the biological contamination is selected from the group consisting of food residues, microorganisms, and a combination of food residues and microorganisms.
 37. The method of claim 1, wherein the biological contamination is selected from the group consisting of adenosine triphosphate, adenosine diphosphate, bioflims, carbohydrates, microbes, and phosphatase.
 38. A method for monitoring the hygiene of a surface desired to be free of the presence of biological contamination, which method comprises the steps of: a) obtaining a sample from said surface; b) adding to said sample a hydrolytic enzyme for converting a carbohydrate that contains glucose to glucose, said hydrolytic enzyme comprising invertase; and c) detecting the presence or absence of glucose that has been enzymatically derived in step b from said sample, wherein the presence of said derived glucose from said sample is an indicator of said contamination on said surface.
 39. The method of claim 38 wherein less than 10 minutes elapses between step b and step c.
 40. The method of claim 38 wherein less than 5 minutes elapses between step b and step c.
 41. The method of claim 38, further comprising adding to said sample a phosphorylase enzyme and a carbohydrate substrate to produce, in the presence of a phosphate in said sample, a reaction product of glucose and a phosphate-bound saccharide reaction product.
 42. A method for monitoring the hygiene of a surface desired to be free of the presence of biological contamination, which method comprises the steps of: a) obtaining a sample from said surface; b) adding to said sample a hydrolytic enzyme for converting a carbohydrate that contains glucose to glucose, said hydrolytic enzyme comprising sucrase; and c) detecting the presence or absence of glucose that has been derived in step b from said sample, wherein the presence of the glucose derived in step b from said sample is an indicator of said contamination on said surface.
 43. The method of claim 42 wherein less than 10 minutes elapses between step b and step c.
 44. The method of claim 42 wherein less than 5 minutes elapses between step b and step c.
 45. The method of claim 43, further comprising adding to said sample a phosphorylase enzyme and a carbohydrate substrate to produce, in the presence of a phosphate in said sample, a reaction product of glucose and a phosphate-bound saccharide reaction product.
 46. A method for monitoring the hygiene of a surface desired to be free of the presence of biological contamination, which method comprises the steps of: a) obtaining a sample from said surface; b) adding to said sample a hydrolytic enzyme for converting a carbohydrate that contains glucose to glucose, said hydrolytic enzyme comprising beta-galactosidase; and c) detecting the presence or absence of glucose that has been derived in step b from said sample, wherein the presence of the glucose derived in step b from said sample is an indicator of said contamination on said surface.
 47. The method of claim 46 wherein less than 10 minutes elapses between step b and step c.
 48. The method of claim 46 wherein less than 5 minutes elapses between step b and step c.
 49. The method of claim 46, further comprising adding to said sample a phosphorylase enzyme and a carbohydrate substrate to produce, in the presence of a phosphate in said sample, a reaction product of glucose and a phosphate-bound saccharide reaction product.
 50. A method for monitoring the hygiene of a surface desired to be free of the presence of biological contamination, which method comprises the steps of: a) obtaining a sample from said surface; b) adding to said sample a hydrolytic enzyme for converting a carbohydrate that contains glucose to glucose, said hydrolytic enzyme comprising amylase; and c) detecting the presence or absence of glucose that has been derived in step b from said sample, wherein the presence of the glucose derived in step b from said sample is an indicator of said contamination on said surface.
 51. The method of claim 50 wherein less than 10 minutes elapses between step b and step c.
 52. The method of claim 50 wherein less than 5 minutes elapses between step b and step c.
 53. The method of claim 50, further comprising adding to said sample a phosphorylase enzyme and a carbohydrate substrate to produce, in the presence of a phosphate in said sample, a reaction product of glucose and a phosphate-bound saccharide reaction product.
 54. A method for monitoring the hygiene of a surface desired to be free of the presence of biological contamination, which method comprises the steps of: a) obtaining a sample from said surface; b) adding to said sample a hydrolytic enzyme for converting a carbohydrate that contains glucose to glucose, said hydrolytic enzyme comprising cellulase; and c) detecting the presence or absence of glucose that has been derived in step b from said sample, wherein the presence of the glucose derived in step b from said sample is an indicator of said contamination on said surface.
 55. The method of claim 54 wherein less than 10 minutes elapses between step b and step c.
 56. The method of claim 54 wherein less than 5 minutes elapses between step b and step c.
 57. The method of claim 54, further comprising adding to said sample a phosphorylase enzyme and a carbohydrate substrate to produce, in the presence of a phosphate in said sample, a reaction product of glucose and a phosphate-bound saccharide reaction product.
 58. A method for monitoring the hygiene of a surface desired to be free of the presence of biological contamination, which method comprises the steps of: a) obtaining a sample from said surface; b) introducing the sample onto a capillary-flow membrane containing reagents for production of glucose and components for colorimetric detection of glucose; and c) detecting the presence or absence of glucose that has been produced in step b from said sample, wherein the presence of the glucose produced in step b from said sample is an indicator of said contamination on said surface.
 59. The method of claim 58 wherein said reagents comprise a hydrolytic enzyme.
 60. The method of claim 58 wherein said reagents for production of glucose is a hydrolytic enzyme selected from the group consisting of amylase, invertase, beta-galactosidase, sucrase and cellulase.
 61. The method of claim 58 wherein less than 10 minutes elapses between step b and step c.
 62. The method of claim 58, further comprising adding to said sample a phosphorylase enzyme and a carbohydrate substrate to produce, in the presence of a phosphate in said sample, a reaction product of glucose and a phosphate-bound saccharide reaction product. 